About 6% of genes in the human genome encode transcription factors (TF) which are the second largest category of proteins encoded by the human genome. Transcription factors play important roles in the regulation of gene expression and also are key nodes for intracellular signaling network, wherein, various signaling pathways triggered by intracellular and extracellular stimulations are cross linked with each other via transcription factors. Thus, transcription factors and their complexes have been attracting great concern. However, due to their low-abundance expression level (only accounting for 0.01-0.001% of total proteins within cells), it is very difficult to purify and identify transcription factors and complexes at protein level. Purification of transcription factors by a conventional chromatography method usually needs hundreds of liters of cell cultures. However, the yielded protein which has been enriched 10,000-100,000 times is barely enough for chemical and functional analysis. While antibodies are undoubtedly the best affinity reagents for detecting proteins, in fact, the commercial available antibodies with high and specific affinity to certain transcription factors are limited. Furthermore, the generation of usable antibodies for detecting endogenous proteins is a process of trial and error. Thus, applications of antibodies to affinity purification of endogenous transcription factors and their complexes are limited. To date, only less than 5% of transcription factors have been purified and identified. Therefore, a method for the purification and identification of the entire family of endogenous transcription factors is in great need.
Transcription factors regulate gene expression by binding to DNA cis-elements located in the regulatory region of target genes. Transcription factors include general transcription factors (e.g., subunits of general transcription factor II (TF II) complex, TATA-binding protein and etc.) and specific transcription factors (such as Spl, C/EBP, AP1 and etc.). During transcription, specific transcription factors bind to promoters and general transcription factors are recruited to the DNA sequence at 40˜60 base pairs upstream or downstream from the transcriptional initiation site, initiating the synthesis of RNA. Recently, more and more researches have found that structural properties of DNA binding elements will affect the formation of the transcriptional initiating complex. In other words, the nucleic acid composition of the transcription factor binding site will affect the recruitment of its co-regulators, which to a certain extent determine whether the transcription factor will act as an activator or a repressor of target genes. For this reason, it is very important to develop a method for isolation and identification of endogenous transcription factors and their complexes. The applications of such methods will shed light on the understanding of the transcriptional network of target genes.
Endogenous protein levels of transcription factors are usually very low and it is difficult to analyze the expression profile of transcription factors on proteome scale by conventional methods. To profile the endogenous transcription factors in cells or tissues, it is necessary to enrich and isolate the transcription factors by affinity purification strategy using specific reagents (such as antibodies). However, limited types of antibodies and the high cost constrained the affinity purification of endogenous transcription factors by using antibodies. In addition, only a few transcription factors can be analyzed by antibody affinity purification in a single experiment. It is hard to enrich and identify most of transcription factors expressed in certain cells or tissues by this strategy.